Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.